Osteoporosis is estimated to affect over half of adults in the United States over the age of 50, and 1 in 3 women over the age of 80 will suffer a hip fracture; of those who fracture, nearly 15-20% die within 1 year. Overall, the costs associated with osteoporosis in the United States alone were estimated at $19 billion in 2005. This project involves the design, development and evaluation of an new molecular imaging radiotracers for Positron Emission Tomography (PET) studies of osteoporosis and other musculoskeletal diseases (e.g., osteoarthritis, rheumatoid arthritis, osteoporosis) involving potential bone loss. The project will develop radiolabeled forms of known high affinity inhibitors of cathepsin K, a cyteine protease that in highly expressed in osteoclasts and that is responsible for type I collagen degradation and bone resorption. The potential for in vivo imaging of cathepsin K has been demonstrated using optical methods, and this project will extend this concept to a clinically translatable imaging method using radionuclide labeling. This grant will prepare series of radiolabeled inhibitors based on the published cyanopyrimidine and pyrrolopyrimidine scaffolds, using isotopic (carbon-11) substitution or synthesis of fluorinated analogs (for fluorine-18 labeling). The compounds selected as initial targets for carbon-11 labeling all have high affinity for human cathepsin K (< 10 nanomolar), excellent selectivity over cathepsins L and S (100-1000 fold selective) and all are readily labeled using [11C]methyl iodide. New fluorinated analogs where fluoroethyl groups replace methyl groups will be evaluated in vitro for inhibitory action on cathepsin K enzymatic activity, using a fluorescent assay, and potent (<10 nM) inhibitors labeled using analogous [18F]fluoroalkylation reactions. In vivo proof of concept studies to demonstrate osteoclast-dependent localization of radiotracers will be done in rats using focal skeletal injections of RANKL (receptor activator for nuclear facktorB ligand, to induce osteoclastogenesis) or osteoprotogerin (to inhibit osteoclastogenesis). Extent of bone loss will be monitored by microCT. Verification that radioactivity localization is cathepsin-specific will be done in RANKL treated rats and blocking with cold doses of enzyme inhibitor. Successful radiotracers that exhibit high uptake and retention in RANKL-treated animals that is blocked by cold inhibitor, and reduced uptake in osteoprotegrin-treated animals, can then be further evaluated as potential radioligands for human imaging. Detection of increased osteoclastic cellular activity may provide new diagnostic criteria that take osteoclast activity into account in addition to standard measures of structural integrity, and thus provide immediate feedback on the efficacy of a chosen treatment protocol before waiting for further downstream gain or loss of bone to occur. .